The possible role of protein kinase C in the regulation of glucose transport in the rat adipose cell has been examined. This study suggests that the mechanisms through which insulin and PMA stimulate glucose transport are distinct but interactive. A new impermeant photoaffinity label has been used for identifying cell surface glucose transporters in isolated rat adipose cells. The results show that while GLUT4 is the major glucose transporter isoform under all conditions, it is selectively and markedly enriched in response to insulin but not PMA which increases GLUT1 and GLUT4 equally. Furthermore, stimulation of glucose transport activity correlates closely with the appearance of GLUT4 on the cell surface in response to both insulin and PMA but does not correlate with the sum of GLUT1 and GLUT4 appearance. The effects of fluorescein isothiocyanate II (FITC) on the actions of insulin in rat adipocytes have been studied. It is concluded that FITC at pH 9.0 (a) renders both glucose transport and phosphodiesterase activities less insulin sensitive presumably by modifying the cellular hormone receptor, and (b) makes glucose transport activity less responsive to insulin presumably by (i) blocking hormone-dependent translocation of glucose transporter and (ii) mildly inhibiting intrinsic glucose transport activity. The subcellular trafficking of tracer-tagged GLUT4 between the plasma membranes and low- density microsomes of rat adipose cells has been studied. Cell-surface GLUT4 have been initially tracer-tagged in the insulin-stimulated state with a [3H]-bis-mannose. The initial exper- iments show that insulin does not alter the half-time for GLUT4 endocytosis but instead increases the rate of exocytosis. Additional data suggest that the cells' entire complement of GLUT4 is involved in the recycling process. Finally, detailed time-course data suggest that there may be plasma membrane intermediate states in the GLUT4 trafficking pathways. Peptides from the alpha 1 domain of the major histocompatibility complex class I antigen (MHC class I) enhance cellular glucose uptake above that of maximal insulin stimulation, prolong the effect of insulin, and inhibit insulin receptor internalization in rat adipose cells. Based on the new data here, we now propose that MHC class I molecules may be involved in regula- tion of the internalization process of cell surface integral membrane pro- teins such as the glucose transporter, IGF-II receptor, and insulin receptor. We have also found that EGF in combination with certain MHC class I-derived peptides is insulinomimetic and that this effect is independent of insulin receptor activity.